Annual Report, 2008[unreadable] [unreadable] [unreadable] 1st Project summary: [unreadable] [unreadable] For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. [unreadable] [unreadable] Our recent studies have been concerned with measuring the internal concentration of polyamines in Saccharomyces cerevisiae under various growth conditions, and, in particular, measuring the internal concentration in auxotrophic mutants grown on limiting spermidine. These studies required modification of our analytical techniques to permit a more sensitive determination of polyamines in cellular extracts. We have found that the internal concentration of spermidine needed for growth is much less (1/1000) than the large amounts normally present in the cells, and that this lower concentration of spermidine is largely used for the biosynthesis of hypusine (40- 50 %), a modification of eIF5A that is critical for protein biosynthesis in all eukaryotes. [unreadable] [unreadable] Currently we have further extended this study to find out the minimum concentration of spermidine and hypusinated eIF5A required for both optimum growth and protein synthesis. We have found that the total amount of eIF5A (unmodified plus hypusinated) is not affected by the internal concentration of spermidine, but when the spermidine concentration is limiting, only 5 % of the eIF5A is hypusinated. Since both growth and protein synthesis were near normal , these data indicate that this amount of modified eIF5A is sufficient for growth and protein synthesis.. A number of polyamine analogs were also tested for their ability to support growth of the polyamine auxotrophs in the absence of spermidine. The importance of hypusine modification for cell growth is further supported by finding that, of the various analogs tested, only 1-methyl spermidine and its S-sterioisomer, supported growth; these two analogues were the only ones that had been shown in in vitro studies to act as a substrate for the modification of eIF5A. Overall our results suggest that hypusine modification of eIF5A is a most important and essential function for spermidine in supporting the growth of S. cerevisiae polyamine auxotrophs. This conclusion is of special interest not only in the polyamine and eIF5A field but also in the protein translation field.[unreadable] We have also been continuing our studies on the role of polyamines in eubacteria (Escherichia coli). The eubacteria lack any spermidine modified hypusinated protein. In our previous studies we have found that polyamine deficient mutants of E. coli grow at 30-40% of the growth rate of a polyamine supplemented culture and are sensitive to oxidative stress. However, using our more sensitive HPLC analytical system (above) we find that these mutants contain trace amount of putrescine and significant amount of cadaverine. Thus, for a better test of the requirements of polyamines in E. coli we have now developed a series of deletion mutants in different polyamine biosynthetic pathways (e.g. cadA, lysB, speF, etc) and constructed a mutant that has deletions of all of the polyamine biosynthetic genes. This strain does not contain even trace amount of any polyamines, but still grows at 50-60% of the normal growth rate in polyamine deficient medium. This result is extremely surprising, as this is in contrast to the reports from various laboratories on the effects of polyamines in in vitro studies in E. coli. Our growth studies (above) were carried out in air. In contrast we found that the polyamine-deficient cells die when grown in 95% oxygen/5% carbon dioxide.[unreadable] [unreadable] Recently we made a surprising observation that, even though the polyamine-free cells grew well in air, they did not grow under anaerobic conditions unless polyamines were added to the medium. We are currently investigating the possible reason(s) for this unexpected result by using phenotypic microarray techniques. [unreadable] [unreadable] We have recently started a new project in the C. elegans model system in collaboration with Dr. Kevin OConnell in our laboratory. In this project we have exploited the use of the newly developed E. coli polyamine auxotrophs as a food source for C.elegans polyamine mutants and to study the polyamine requirements of this organism. Preliminary studies have shown developmental defect and embryonic lethality in C. elegans during different developmental stages in the absence of polyamines.